Exogenous EPEC polysaccharide protects against HD-5 killing.

<p>Survival of EPEC ∆<i>gfcA</i>∆<i>waaL</i> cells in the presence of 5 mM HD-5 supplemented with the indicated amount of EPEC polysaccharide purified from wild-type cells. The control does not have exogenous polysaccharide added to the sample. Results are expressed as means ± SEs of triplicate samples. Data shown are representative of at least three independent experiments. Asterisks indicate statistical significance compared to the control group; **, <i>P</i> <0.01; ****, <i>P</i> <0.0001 by two-way ANOVA and Bonferroni’s multiple comparison <i>post </i><i>hoc</i> test.</p

Expression of surface structures by EPEC.

<p>Transcription of the indicated genes was quantified by qPCR. Data shown (2^-ΔCT x 10³) are normalized against transcription of the 16S RNA gene. Results are expressed as means ± SEs of triplicate samples. Asterisks indicate statistical significance; ***, <i>P</i> <0.001 by one-way ANOVA and Bonferroni’s multiple comparison <i>post </i><i>hoc</i> test.</p

Interplay between G4C and O-antigen.

<p>(A) Silver staining of proteinase K-treated LPS of the indicated EPEC strains. All samples were normalized (by OD₆₀₀) to ensure that the same number of cells was used. Data shown are representative of three independent experiments. (B) Percent of total combined hexose and hexosamine in each purified polysaccharide preparation ([mg hexose +hexosamine]/ mg purified polysaccharide) from the indicated strain. Results are expressed as mean ± SDs of samples. Asterisks indicate statistical significance; **, <i>P</i><0.01; ****, <i>P</i><0.0001 by one-way ANOVA and Bonferroni’s <i>post </i><i>hoc</i> comparison test. </p

Capsule production and HD-5 resistance in EPEC and EHEC.

<p>(A) Transcription of <i>gfcA</i> in the EPEC and EHEC wild-type strains was quantified by qPCR. Data shown (2^-ΔCT x 10³) are representative of <i>gfcA</i> gene expression normalized against 16S RNA gene expression. Results are expressed as means ± SEs of triplicate samples. Asterisks indicate statistical significance; **, <i>P</i> <0.01 by paired <i>t</i> test. (B) Capsule staining of EPEC and EHEC wild-type strains, capsules are visualized by negative staining at a magnification of 100 X. Images shown are representative of at least ten fields of view from three independent experiments. (C) Survival of EPEC and EHEC wild-type cells in the presence of 5 µM HD-5. Results are expressed as means ± SEs of triplicate samples. Data shown are representative of at least three independent experiments. Asterisks indicate statistical significance; **, <i>P</i> <0.01 by two-way ANOVA and Bonferroni's multiple comparison <i>post </i><i>hoc</i> test.</p

HD-5 and HD-6 do not synergize.

<p>Survival of EPEC wild-type, ∆<i>gfcA</i>, ∆<i>gfcA</i>(p<i>gfcA</i>) strains (A) and EPEC wild-type, ∆<i>waaL</i>, ∆<i>waaL</i>(p<i>waaL</i>), ∆<i>gfcA</i>∆<i>waaL</i> and ∆<i>gfcA</i>∆<i>waaL</i>(p<i>waaL</i>) strains (B) in the presence of 4 µM HD-6. (C) Survival of EPEC wild-type, ∆<i>gfcA</i>, ∆<i>gfcA</i>(p<i>gfcA</i>), ∆<i>waaL</i> and ∆<i>waaL</i>(p<i>waaL</i>) in the presence of 5 µM HD-5 and 4 µM HD-6. Results are expressed as means ± SEs of triplicate samples. Data shown are representative of at least three independent experiments. Asterisks indicate statistical significance; **, <i>P</i><0.01; ****, <i>P</i> <0.0001 by one-way ANOVA and Bonferroni’s multiple comparison <i>post </i><i>hoc</i> test.</p

The O-antigen promotes resistance to HD-5.

<p>Survival of the indicated EPEC strains in the presence of LL-37 (A) and HD-5 (B and C) at the indicated concentrations. Results are expressed as means ± SEs of triplicate samples. Data shown are representative of at least three independent experiments. Asterisks indicate statistical significance; *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001; ****, <i>P</i> <0.0001 by two-way ANOVA and Bonferroni’s multiple comparison <i>post </i><i>hoc</i> test.</p

Oligonucleotides used in this study.

<p>Oligonucleotides used in this study.</p

Amino acid sequence alignment of <i>A. fumigatus</i> MedA putative NLSs 1–4 with other MedA homologues.

<p>(A) Sequence alignment and motif prediction using PSORT II identified NLS1 sequence among orthologues of MedA. (B) Sequence alignment of the MedA minimal nuclear localization domain, MedA^346–557 with other MedA orthologues. The sequences representing the putative NLSs 2, 3, and 4 are boxed. The basic amino acids within the putative NLSs of <i>A. fumigatus</i> MedA and the corresponding amino acids in MedA orthologues are highlighted in gray. The presence of an asterisk or a colon below the basic amino acids indicates a fully or strongly conserved residue, respectively. Numbers indicate the amino acid position within the primary amino acid sequence of the protein. Af_MedA: <i>A. fumigatus</i> MedA (GenBank: EAL93620.1), An_MedA: <i>A. nidulans</i> MedA (GenBank: AAC31205.1), Nc_ACON-3: <i>N. crassa</i> ACON-3 (GenBank: ADL28820.1), Mg_Acr1: <i>M. grisea</i> Acr1 (GenBank: BAC41196.1), and Fo_Ren1: <i>F. oxysporum</i> Ren1 (GenBank: BAC55015.1).</p

Phenotypic analysis of Δ<i>medA</i> strain expressing MedA^346–557 domain.

<p>(A) Conidia hydrophobicity and biofilm formation of the indicated strains. (B) Survival assay of <i>G. mellonella</i> larvae. 28 worms/strain were infected with 10⁵ swollen conidia. Af293 indicates the <i>A. fumigatus</i> wild type strain; MedA^346–557 indicates expression of this construct under the control of the <i>medA(p)</i> in the Δ<i>medA</i> strain. Analysis of survival data was performed using the log rank test. Statistically significant differences are indicated by asterisk (P value ≤0.05).</p

Relative gene expression of cytoplasmic and nuclear <i>medA</i> measured by RT-PCR.

<p>Af293 is the <i>A. fumigatus</i> wild type strain; MedA, MedA^ΔNLS1, MedA^ΔNLS2, MedA^ΔNLS3, and MedA^ΔNLS4 indicate expression of the corresponding construct under the control of the <i>medA(p)</i> in the Δ<i>medA</i> strain, normalized to <i>medA</i> expression in strain Af293. Error bars represent the standard error of three triplicates for every strain.</p